Malaria still afflicts about half of the world population causing more than 400,000 deaths, mostly children. The global economic toll of malaria is enormous. Most of the drugs that are currently utilized for malaria treatment, including artemisinin-based combination treatments are losing their effectiveness due to widespread emergence of drug resistance. To address the fragility of malaria therapy, we propose to discover novel antimalarial compounds through screening of a library of fungal secondary metabolites. We hypothesize that fungal secondary metabolites, which are underexplored for antimalarial discovery, will provide us with a unique opportunity to investigate medicinally relevant but untapped chemical space for the discovery of novel malaria therapeutics. Premise for this proposal is based on our promising preliminary screen that has identified fungal extracts and pure compounds of fungal origin with potent antiplasmodial activities. To prove this hypothesis, we propose herein to (1) Screen a library of 10,000 extracts derived from diverse fungal species and dereplicate prioritized bioactive extracts to identify and determine structures of selective antiplasmodial compounds that are active against multiple parasite developmental stages; hits will be screened to determine cross-resistance, and killing rate. (2) Active compounds will be prioritized by in vitro physicochemical, in vivo pharmacology and in vivo efficacy studies. (3) Target identification of prioritized hits will be determined by in vitro evolution of resistance followed by whole genome sequencing. Target validation will be conducted by CRISPR/Cas9 mediated genome editing. The research in this endeavor will be conducted through a multidisciplinary collaboration between the laboratories of Debopam Chakrabarti (University of Central Florida), Robert Cichewicz (University of Oklahoma), Kirsten Hanson (University of Texas San Antonio), Elizabeth Winzeler and Jeremiah Momper (University of California San Diego) with combined expertise in natural product chemistry, malaria cell biology, anti-infective discovery, target identification, and validation. This is a highly significant endeavor, as we will discover novel lead compounds with validated targets for therapy against multidrug resistant malaria.